Many eukaryotic proteins function in multi-subunit complexes with a stoichiometry that needs to be strictly maintained. It is thought that the degradation of unassembled subunits might be an essential mechanism that controls complex stoichiometry. Elimination of unassembled proteins is also crucial for protein homeostasis because unassembled proteins often contain exposed hydrophobic segments that can lead to protein aggregation and cytotoxicity. In fact, a major effort in developing anti-cancer therapies that target proteostasis-addicted tumors is based on the assumption that unbalanced protein assembly due to aneuploidy or other genome instabilities in cancer cells render them more susceptible to chemicals that disturb the proteostasis network. In this regard, identification of cellular components mediating the degradation of unassembled proteins may reveal novel anti-cancer targets. Membrane and secreted protein complexes are usually assembled in the endoplasmic reticulum (ER) after individual subunits have been imported into the ER. The assembly process is subject to a strict checkpoint regulation enforced by an efficient protein quality control (PQC) mechanism. The ER PQC pathway employs chaperones, lectins and other enzymes to monitor the assembly process, identifying unassembled polypeptides for retrotranslocation, ubiquitination and proteasomal degradation in the cytosol. This conserved process is termed ER-associated protein degradation (ERAD), which is critical for cell viability because unassembled polypeptides can interfere with normal protein assembly when they become misfolded or form non-specific interactions. Unassembled ER proteins can also co-aggregate with essential cellular factors such as chaperones to cause ER stress, which if not rectified, can lead to cell death. Many proteins in the cytosol and nucleus also form multi-subunit assemblies, but the mechanism by which cells eliminate unassembled soluble proteins is not well understood. Several studies have investigated the mechanism of cytoplasmic and nuclear PQC, which remove misfolded or damaged proteins from the cytoplasm and nucleus, respectively. These studies identified several pathways that target misfolded proteins of different classes to the proteasome for degradation. For example, the ribosome-associated ubiquitin ligase Ltn1 in budding yeast recognizes and ubiquitinates defective translation products due to non-stop messenger RNAs. In mammalian cells, a chaperone-associated ubiquitin ligase named RNF126 targets mislocalized membrane proteins for degradation. However, these studies did not use substrates representing unassembled polypeptides. Therefore, it is unclear whether these cytosolic PQC pathways play a role in unassembled soluble protein degradation (USPD). To date, the best-characterized cytosolic quality control pathway is the N-end rule pathway, which mediates the degradation of substrates bearing an N-terminal destabilizing element termed degron. The N-end rule substrates have been classified into three major groups: those with an N-terminal destabilizing residue, those with an exposed acetylated N-terminal methionine residue and a group of proteins with an N-terminal initiator methionine followed by a hydrophobic residue. A major ubiquitin ligase responsible for degradation of non-acetylated N-end rule substrates is UBR1 and the related enzymes UBR2 and UBR3. In yeast, a protein named CNOT4 was recently identified as the ubiquitin ligase for an unassembled soluble protein carrying an exposed acetylated N-terminal methionine. It is conceivable that some USPD substrates may carry one of the above-mentioned degrons, but for those without a predicted N-end rule degron, how they are targeted for degradation is unclear. We have established model substrates to study N-end rule independent USPD in mammalian cells. Our study establishes HUWE1 as an enzyme that ubiquitinates substrates bearing exposed hydrophobic residues due to incomplete assembly to cause their degradation by the proteasome. We identify endogenous HUWE1 substrates, which reveal a new surveillance system that safeguards the proteostasis network of the eukaryotic cells.